PROJECT SUMMARY First-line chemotherapy is the standard of care for patients with triple-negative breast cancer (TNBC). While short-term response is achievable, most patients succumb to recurrence due to metastasis. Micrometastasis encompasses a small population of dormant disseminated tumor cells (dDTCs) that survive in quiescent/senescent states prior to initiating their ?explosive? metastatic outgrowth. Standard chemotherapy is completely ineffective against the slow-dividing dDTCs. In contrast, cancer immunotherapy is based on the premise of immune-recognition and targeted killing of tumor cells, thus possess the promising power to control dormant metastatic cancer cells. However, one major hurdle in immunotherapy is to overcome the profound immunosuppression within the tumor microenvironment (TME). TME is associated with the accumulation of dysfunctional antigen-presenting cells (APCs). An effective approach to alter TME is to reprogram these inhibitory APCs into properly activated APCs that stimulate tumor antigen-specific T cells. We designed an immuno-stimulatory nanoparticle that exploits the unique physiological features of metastatic TME, which allows the systemic delivery of nanoparticles to achieve a robust immunostimulation within the TME. First, to drive a sustainable antitumor immune response, we harness two synergistic innate immune pathways by co- delivering two immune agonists. The immuno-NP is co-loaded with an agonist of the Stimulator of Interferon Genes (STING) pathway and a Toll-like receptor 4 (TLR4) agonist, which synergize to produce high levels of Type I interferon (IFN) ?. The dual-agonist NP guarantees uptake of both agonists by the same APC, which elicits functional synergy. Second, the immuno-NP facilitates proficient presentation of each agonist to the appropriate intracellular location of APCs. Third, the immuno-NP is designed for systemic administration targeting the APC-rich perivascular areas of metastasis, leading to uptake predominantly by APC cells. As a result, high levels of IFN? produced within the tumor site lead to the activation of APC and NK cells that consequently drive the recruitment of additional immune cells as well as the activation of tumor-reactive cytotoxic CD8+ T cells. Any immuno-NP-associated toxicity was minimal and reversible. Our central hypothesis is that the dual-agonist cargo (STING and TLR4 agonists) of the immuno-NP targeted to the perivascular regions of metastasis will produce a strong IFN?-driven antitumor immune response. Aim 1: Optimize an immuno-NP design that targets the metastatic TME with high efficiency and mediates co- delivery of the dual-agonist cargo at the ratio of STING/TLR4 agonists for optimal functional synergy. Aim 2: Evaluate the short and long-term safety profile of the immuno-NP and characterize the mechanism of antitumor immune responses associated with dosage and frequency of immuno-NP administration. Aim 3: Evaluate the therapeutic efficacy of the immuno-NP as a monotherapy and in combination with immune checkpoint inhibitors in murine models of metastatic TNBC.